WO2024114542A1 - Vaccin à poxvirus chimérique multi-antigène et son utilisation - Google Patents

Vaccin à poxvirus chimérique multi-antigène et son utilisation Download PDF

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WO2024114542A1
WO2024114542A1 PCT/CN2023/134141 CN2023134141W WO2024114542A1 WO 2024114542 A1 WO2024114542 A1 WO 2024114542A1 CN 2023134141 W CN2023134141 W CN 2023134141W WO 2024114542 A1 WO2024114542 A1 WO 2024114542A1
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amino acid
acid sequence
seq
poxvirus
immunogen
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Chinese (zh)
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高福
王奇慧
杜沛
孔天翔
仵丽丽
璩骁
齐建勋
马任义
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中国科学院微生物研究所
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N5/0686—Kidney cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present application belongs to the field of biomedicine, and specifically relates to a poxvirus multi-immunogen chimeric or mixed vaccine and its use.
  • Poxviruses represented by monkeypox virus, are a type of large nucleocytoplasmic DNA virus with a viral genome of approximately 130-375kbp, which can encode up to 200 viral proteins.
  • the virus particles of poxviruses are also relatively complex. They have two different forms of infectious virus particles, called intracellular mature viruses (IMV) and extracellular enveloped viruses (EEV).
  • IMV intracellular mature viruses
  • EEV extracellular enveloped viruses
  • IMV intracellular mature viruses
  • EEV extracellular enveloped viruses
  • IMV intracellular mature viruses
  • EEV extracellular enveloped viruses
  • IMV intracellular mature viruses
  • EEV extracellular enveloped viruses
  • IMV has a layer of capsule, which has higher stability than EEV and is mainly involved in the spread of the virus between hosts.
  • EEV on the other hand, has a special outer membrane structure, which is mainly involved in the spread of the virus in the host. Because IMV and EEV have different membrane structures, membrane components and cell infection mechanisms, their surface neutralizing antigens are also
  • Monkeypox virus is a poxvirus that is highly homologous to smallpox virus. Since its discovery in 1958, it has long been locally transmitted in central and western Africa and has evolved into multiple strains. However, in 2022, MPXV showed a global epidemic trend. As of July 23, it has spread to 75 countries and infected more than 15,000 people. It was declared an "international public health emergency" by the World Health Organization. Although current vaccines have the ability to defend against monkeypox virus, it is worth noting that MPXV has recently been found to have an accelerated mutation frequency and may produce escape mutants.
  • JYNNEOS TM also known as Imvamune or Imvanex
  • Imvanex produced by Ankara-Bavarian Nordic
  • Imvanex produced by Sanofi Pasteur. Both vaccines are attenuated live vaccines originally used to prevent smallpox. It is a second-generation vaccine that has the ability to replicate in the human body. There is a risk of encephalitis, myocarditis, progressive vaccinia infection, etc. after vaccination. It is not suitable for young children, pregnant women, and people with low or impaired immune function.
  • JYNNEOS TM is a third-generation vaccine. Since it cannot replicate in the human body, its safety is improved compared to the first and second generation vaccines, but the immune effect has also been reduced to a certain extent.
  • Live virus vaccines have the following common disadvantages: (1) The virus comes from a live virus and is obtained through attenuation and weakening culture. It retains the virus antigens completely, but there is a possibility of incomplete attenuation or restoration of virulence after mutation; (2) The vaccine contains all the antigens of the virus, has a complex composition, and has a high possibility of causing side effects; (3) The proportion of effective immunogenic components in the vaccine is low, and the immunogenicity produced by the same dose of vaccination is weak; (4) Some components of the virus may have inhibitory effects. The function of host immunity will have a counter-effect and reduce the immunogenicity of the vaccine. Vaccines with clear ingredients (mRNA vaccines/recombinant protein vaccines, etc.) can solve the above problems. However, for complex viruses such as poxviruses, finding key ingredients as immunogens is a major technical bottleneck.
  • the present application provides a poxvirus multi-immunogen chimeric or mixed antigen, wherein the chimeric or mixed antigen comprises:
  • Immunogen I monkeypox virus A35R protein or an antigenic fragment thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity thereto;
  • Immunogen II Monkeypox virus M1R protein or an antigenic fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto; and
  • Immunogen III Monkeypox virus B6R protein or an antigenic fragment thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity thereto.
  • the antigenic fragment of the A35R protein is the extracellular segment of the protein or a portion thereof, preferably having an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence as shown in SEQ ID NO: 1 plus an amino acid sequence of a fragment extending 1-30 amino acids from the amino acid sequence to the N-terminus of the A35R protein, preferably a peptide segment of the amino acid sequence as shown in SEQ ID NO: 2;
  • the antigenic fragment of the M1R protein is the extracellular segment of the protein or a part thereof, preferably a peptide segment having an amino acid sequence as shown in SEQ ID NO: 3;
  • the antigenic fragment of the B6R protein is the extracellular segment of the protein or a part thereof, preferably a peptide segment having an amino acid sequence as shown in SEQ ID NO:4.
  • the poxvirus multi-immunogen chimeric or mixed antigen is a multi-immunogen mixed antigen.
  • the mixed antigen is a mixture of immunogens I, II and III;
  • the immunogen I has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and having the same or substantially the same immunogenicity as the amino acid sequence;
  • the immunogen II has an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 3, and having the same or substantially the same immunogenicity as the amino acid sequence;
  • the immunogen III has an amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence having the same or substantially the same immunogenicity as the amino acid sequence as shown in SEQ ID NO:4 obtained by substituting, deleting or adding one or more amino acids, preferably an amino acid sequence as shown in SEQ ID NO:5;
  • the mass ratio of the immunogens I, II and III is 1:1:1.
  • the poxvirus multi-immunogen chimeric or mixed antigen is a multi-immunogen chimeric antigen
  • the chimeric antigen is a single chain formed by one or more immunogens I, II, and III in series.
  • the chimeric antigen comprises an amino acid sequence arranged in the pattern of A1-C 1 -MC 2 -A2-C 3 -B, wherein:
  • A1 represents the monkeypox virus A35R protein or antigenic fragment I thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto,
  • A2 represents the monkeypox virus A35R protein or antigenic fragment II thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto,
  • M represents the monkeypox virus M1R protein or an antigenic fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto,
  • B represents monkeypox virus B6R protein or an antigenic fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto,
  • C 1 , C 2 , and C 3 are each independently none or a linking sequence (GGGGS)m, wherein m is any integer between 1 and 10; and
  • A1 and A2 are the same or different.
  • A1 represents the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 1, and having the same or substantially the same immunogenicity as the amino acid sequence;
  • A2 represents the amino acid sequence as shown in SEQ ID NO:1, or the amino acid sequence as shown in SEQ ID NO:1 plus the amino acid sequence of a fragment extending 1-30 amino acids therefrom to the N-terminus of the A35R protein, preferably the amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the above amino acid sequence and having the same or substantially the same immunogenicity as the above amino acid sequence; wherein the amino acid sequence
  • the A35R peptide segment with the amino acid sequence shown in SEQ ID NO: 1 is a functional peptide segment, and the segment extending 1-30 amino acids to the N-terminus thereof acts as a linker;
  • M represents the amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO:3, and having the same or substantially the same immunogenicity as the amino acid sequence;
  • B represents an amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence obtained by substituting, deleting or adding one or several amino acids to the amino acid sequence as shown in SEQ ID NO:4, and having the same or substantially the same immunogenicity as the amino acid sequence, preferably an amino acid sequence as shown in SEQ ID NO:5.
  • A1 represents the amino acid sequence shown in SEQ ID NO: 1
  • A2 represents the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2
  • M represents the amino acid sequence shown in SEQ ID NO: 3
  • B represents the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5;
  • A1 represents the amino acid sequence shown in SEQ ID NO: 1
  • A2 represents the amino acid sequence shown in SEQ ID NO: 2
  • M represents the amino acid sequence shown in SEQ ID NO: 3
  • B represents the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5;
  • C 1 , C 2 , and C 3 are all absent;
  • the chimeric antigen comprises an amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:7.
  • the chimeric antigen comprises an amino acid sequence arranged in the pattern of AC 1 '-MC 2 '-B, wherein:
  • A represents monkeypox virus A35R protein or an antigenic fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto,
  • M represents the monkeypox virus M1R protein or an antigenic fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto,
  • B represents monkeypox virus B6R protein or an antigenic fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity thereto,
  • C 1 ' and C 2 ' are each independently none or a linker sequence (GGGGS)n, wherein n is any integer between 1-10.
  • A represents an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 1, and having the same or substantially the same immunogenicity as the amino acid sequence;
  • M represents the amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 3, and having the same or substantially the same immunogenicity as that of the amino acid sequence;
  • B represents an amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence obtained by substituting, deleting or adding one or several amino acids to the amino acid sequence as shown in SEQ ID NO:4, and having the same or substantially the same immunogenicity as the amino acid sequence, preferably an amino acid sequence as shown in SEQ ID NO:5.
  • SEQ ID NO:5 contains only one C140S mutation; after this mutation, the number of C (cysteine) on the surface of the B6R peptide segment changes from an odd number to an even number, reducing the probability of the protein forming polymers. This is because: the two cysteines on the peptide chain can form a disulfide bond. If there are an even number of Cs on the peptide chain, it will tend to form an intrachain disulfide bond; if there are an odd number of Cs on the peptide chain, the extra C will combine with the extra Cs on other peptide chains to form polymers.
  • A represents the amino acid sequence shown in SEQ ID NO: 1
  • M represents the amino acid sequence shown in SEQ ID NO: 3
  • B represents the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5;
  • both C 1 ' and C 2 ' are absent;
  • the chimeric antigen comprises an amino acid sequence as shown in SEQ ID NO:8 or as shown in SEQ ID NO:9.
  • the N-terminus of the chimeric antigen also contains a signal peptide sequence; optionally, the signal peptide sequence is as shown in SEQ ID NO:19.
  • a multi-immunogen chimeric or mixed antigen is designed that contains the neutralizing antigens A35R and B6R of EEV and the neutralizing antigen M1R of IMV.
  • These three neutralizing antigens are encoded by the A35R gene, B6R gene and M1R gene of monkeypox virus, respectively, and are homologous genes to vaccinia virus A33R, B5R and L1R.
  • the present application provides a method for preparing a poxvirus multi-immunogen chimeric antigen as described in the first aspect above, comprising the following steps:
  • the Kozak sequence and the coding sequence of the signal peptide are added to the 5' end of the nucleotide sequence encoding the poxvirus multi-immunogen chimeric antigen as described in the first aspect above, and the coding sequence of the histidine tag and the stop codon are added to the 3' end. Cloning and expression are carried out, and the correct recombinants are screened. They are then transfected into expression system cells for expression, and the cell culture supernatant is collected to isolate the chimeric antigen.
  • the cells of the expression system are mammalian cells, insect cells, yeast cells or bacterial cells;
  • the mammalian cell is a HEK293T cell, a 293F series cell or a CHO cell; further optionally, the 293F series cell is a HEK293F cell, a Freestyle293F cell or an Expi293F cell;
  • the insect cell is a sf9 cell, a Hi5 cell, a sf21 cell or a S2 cell;
  • the yeast cell is a Pichia pastoris cell or a yeast cell transformed therefrom;
  • the bacterial cells are Escherichia coli cells.
  • the present application provides a polynucleotide encoding the poxvirus multi-immunogen chimeric or mixed antigen as described in the first aspect above.
  • the polynucleotide is a nucleotide sequence optimized for human codons, which may be DNA or mRNA;
  • the polynucleotide is a DNA molecule comprising a DNA sequence as shown in one of SEQ ID NO: 10, 11, 12 or 13, or consisting of the DNA sequence; or, the polynucleotide is an mRNA molecule comprising an mRNA sequence as shown in one of SEQ ID NO: 14, 15, 16 or 17, or consisting of the mRNA sequence;
  • the polynucleotide is a polynucleotide group, which includes or consists of the following DNA molecules: a DNA molecule comprising a DNA sequence as shown in SEQ ID NO:22, a DNA molecule comprising a DNA sequence as shown in SEQ ID NO:23, and a DNA molecule comprising a DNA sequence as shown in SEQ ID NO:24; or, the polynucleotide group includes or consists of the following mRNA molecules: an mRNA molecule comprising an mRNA sequence as shown in SEQ ID NO:25, an mRNA molecule comprising an mRNA sequence as shown in SEQ ID NO:26, and an mRNA molecule comprising an mRNA sequence as shown in SEQ ID NO:27.
  • the present application provides a nucleic acid construct, which comprises the polynucleotide as described in the third aspect above, and optionally, at least one expression control element operably linked to the polynucleotide.
  • the present application provides an expression vector comprising the nucleic acid construct as described in the fourth aspect above.
  • the present application provides a host cell, which is transformed or transfected with the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, or the expression vector as described in the fifth aspect.
  • the present application provides the use of the poxvirus multi-immunogen chimeric or mixed antigen as described in the first aspect, the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, the expression vector as described in the fifth aspect, or the host cell as described in the sixth aspect in the preparation of a drug for preventing and/or treating poxvirus infection;
  • the poxvirus is selected from the group consisting of: monkeypox virus, smallpox virus, cowpox virus and/or vaccinia virus;
  • the drug is a vaccine, such as a DNA vaccine, mRNA vaccine or recombinant protein vaccine based on a eukaryotic expression vector or a viral vector, preferably an mRNA vaccine;
  • the vaccine is in the form of a nasal spray, oral formulation, suppository or parenteral formulation;
  • the nasal spray is selected from aerosols, sprays and powder sprays;
  • the oral preparation is selected from tablets, powders, pills, granules, soft/hard capsules, film-coated agents and pastes; further preferably, the tablets are sublingual tablets; further preferably, the granules are fine granules; further preferably, the powders are powders; further preferably, the pills are pellets;
  • the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid preparation, or an injectable preparation; further preferably, the injectable preparation is a pushable preparation.
  • the present application provides a vaccine or immunogenic composition
  • a vaccine or immunogenic composition comprising the vaccine or immunogenic composition as described in the first aspect above.
  • the vaccine or immunogenic composition is a poxvirus recombinant protein vaccine, which comprises the poxvirus multi-immunogen chimeric or mixed antigen and an adjuvant as described in the first aspect above;
  • the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.
  • the vaccine or immunogenic composition is a poxvirus DNA vaccine comprising:
  • the DNA sequence constructed into the eukaryotic expression vector is a DNA sequence as shown in one of SEQ ID NO: 10, 11, 12 or 13;
  • the DNA vaccine comprises three eukaryotic expression constructs, and the DNA sequence in each eukaryotic expression construct is shown in SEQ ID NO: 22, 23, and 24 respectively;
  • the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.
  • the vaccine or immunogenic composition is a poxvirus mRNA vaccine, and the mRNA vaccine comprises:
  • the mRNA molecule when it is a poxvirus multi-immunogen chimeric antigen, has an mRNA sequence as shown in one of SEQ ID NO: 14, 15, 16 or 17;
  • the mRNA vaccine when it is a poxvirus multi-immunogen mixed antigen, comprises three lipid nanoparticle-packaged mRNA molecules, which have the mRNA sequences shown in SEQ ID NO: 25, 26, and 27, respectively.
  • the vaccine or immunogenic composition is a poxvirus-viral vector vaccine, which comprises:
  • the DNA sequence constructed into the viral backbone vector is a DNA sequence as shown in one of SEQ ID NO: 10, 11, 12 or 13;
  • the poxvirus-viral vector vaccine comprises three viral vector constructs, and the DNA sequence in each viral vector construct is shown as SEQ ID NO: 22, 23, and 24, respectively.
  • the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.
  • the vaccine or immunogenic composition is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation;
  • the nasal spray is selected from aerosols, sprays and powder sprays;
  • the oral preparation is selected from tablets, powders, pills, granules, soft/hard capsules, film coatings and ointments;
  • the tablet is a sublingual tablet
  • the granules are fine granules
  • the powder is a powder
  • the pills are pellets
  • the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid preparation, or an injectable preparation; further preferably, the injectable preparation is a pushable preparation.
  • the present application provides a method for preventing and/or treating poxvirus infection, the method comprising: administering to a subject in need thereof a preventive and/or therapeutically effective amount of the following substances: the multi-immunogen chimeric or mixed antigen as described in the first aspect above, the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, the expression vector as described in the fifth aspect above, the host cell as described in the sixth aspect above, and/or the vaccine or immunogenic composition as described in the eighth aspect above.
  • the "preventively and/or therapeutically effective amount” may vary depending on the subject of administration, the subject organ, symptoms, the method of administration, etc., and can be determined based on the doctor's judgment, taking into account the type of dosage form, the method of administration, the patient's age and weight, the patient's symptoms, etc.
  • the inventors of the present application have designed a multi-immunogen chimeric or mixed antigen against poxvirus (especially monkeypox virus), which comprises three immunogens: (1) monkeypox virus A35R protein or its antigenic fragment (or its derivative peptide segment), (2) monkeypox virus M1R protein or its antigenic fragment (or its derivative peptide segment), and (3) monkeypox virus B6R protein or its antigenic fragment (or its derivative peptide segment); wherein A35R and B6R are neutralizing antigens specific to intracellular mature virus particles (IMV), and M1R is a neutralizing antigen specific to extracellular enveloped virus particles (EEV); a vaccine comprising these three immunogens can stimulate an immune response against two infectious virus particles.
  • monkeypox virus especially monkeypox virus
  • A35R and B6R are neutralizing antigens specific to intracellular mature virus particles (IMV)
  • M1R is a neutralizing antigen specific to extracellular enveloped virus particles (EEV)
  • a vaccine
  • the present application forms a single-chain polypeptide by directly connecting one or more of the monkeypox virus A35R protein or its antigenic fragment, the monkeypox virus M1R protein or its antigenic fragment and the monkeypox virus B6R protein or its antigenic fragment in series or through an appropriate connecting sequence.
  • the resulting single-chain polypeptide not only retains the immunogenicity of each of the three immunogens, but also can more efficiently activate specific protective antibodies against monkeypox virus.
  • the chimeric or mixed antigen vaccine products of the present application have the following advantages:
  • monkeypox virus It has a high specificity for monkeypox virus; existing live virus vaccines are basically developed based on vaccinia virus. Although vaccinia virus and monkeypox virus belong to the same Poxviridae family, there are still certain differences in the neutralizing antigen sequences of the two. Therefore, their protective effect against monkeypox virus has yet to be clarified.
  • the poxvirus vaccine of the present application is developed based on the antigenic epitope of monkeypox virus. Therefore, it has a high specificity for the prevention and treatment of monkeypox virus;
  • Figure 1 shows the expression effect of the chimeric mRNA constructed in Example 1 of the present application detected by Western Blot in cells.
  • Figure 2 is a schematic diagram of the immunization procedure and serum sampling procedure used in Example 2 of the present application.
  • Figure 3 shows the results of specific binding antibody titer detection against A35R, M1R and B6R antigen proteins in mouse sera collected on the 13th day ( Figure 3a) and the 27th day ( Figure 3b) of each chimeric mRNA vaccine immunization procedure as recorded in Example 3 of the present application, detected by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Figure 4 shows the plaque neutralization results of mouse sera collected on the 27th day of the immunization procedure for each chimeric mRNA vaccine tested in Example 4 of the present application.
  • Figure 5 shows the protective effect of each chimeric mRNA vaccine tested in Example 5 of the present application on BALB/c mice challenged with VACV-WR virus intranasally, wherein the horizontal axis shows the number of days after the attack, and the vertical axis shows the percentage of weight change (a) and survival rate (b) of the mice.
  • Figure 6 shows the protective effect of the mixed mRNA vaccine "A/M/B" tested in Example 5 of the present application on BALB/c mice challenged with VACV-WR virus intranasally, wherein the horizontal axis shows the number of days after the attack, and the vertical axis shows the percentage of weight change (a) and survival rate (b) of the mice.
  • Example 1 Antigen design, in vitro preparation, intracellular expression test and lipid nanoparticle packaging of poxvirus multi-immunogen chimeric or mixed mRNA vaccines
  • A35R-M1R-A35R-B6R From N-terminus to C-terminus, the arrangement is A35R-M1R-A35R-B6R, wherein the amino acid sequence of the first A35R peptide from the N-terminus is shown in SEQ ID NO:1, and the amino acid sequence of the second A35R peptide is shown in SEQ ID NO:2; the amino acid sequence of M1R is shown in SEQ ID NO:3; and the amino acid sequence of B6R is shown in SEQ ID NO:4;
  • A35R-M1R-A35R-B6R From N-terminus to C-terminus, the arrangement is A35R-M1R-A35R-B6R’, wherein the amino acid sequence of the first A35R peptide from the N-terminus is shown in SEQ ID NO:1, and the amino acid sequence of the second A35R peptide is shown in SEQ ID NO:2; the amino acid sequence of M1R is shown in SEQ ID NO:3; and the amino acid sequence of B6R’ is shown in SEQ ID NO:5;
  • AMB-C140S From N-terminus to C-terminus, the arrangement is A35R-M1R-B6R’, among which the amino acid sequence of A35R is shown in SEQ ID NO:1; the amino acid sequence of M1R is shown in SEQ ID NO:3; and the amino acid sequence of B6R’ is shown in SEQ ID NO:5.
  • A/M/B a mixed vaccine of A35R, M1R and B6R mRNA vaccines
  • A/M/B a mixed vaccine of A35R, M1R and B6R mRNA vaccines
  • A/M/B mRNA vaccines of single immunogens A35R, M1R and B6R were prepared and packaged respectively, and the three were mixed in a mass ratio of 1:1:1; wherein the single immunogens A35R, M1R and B6R are the A35R peptide segment shown in SEQ ID NO:1, the M1R peptide segment shown in SEQ ID NO:3 and the B6R peptide segment shown in SEQ ID NO:4, respectively.
  • the basic plasmid used for in vitro transcription of mRNA vaccine is pUC57, which is provided by Nanjing GenScript Biotechnology Co., Ltd.
  • DNA expression elements of mRNA vaccine were introduced into the basic plasmid pUC57 by conventional molecular biological methods, including: (1) T7 promoter, (2) DNA coding region of mRNA vaccine (DNA coding sequences of chimeric mRNA vaccines of "AMAB”, “AMAB-C140S” and “AMB-C140S” are shown in SEQ ID NOs: 10, 11 and 13, respectively; DNA coding sequences of single mRNA vaccines of "A35R”, “M1R” and “B6R” are shown in SEQ ID NOs: 22, 23 and 24, respectively), (3) 5' UTR sequence upstream of the coding region (shown in SEQ ID NO: 18), (4) coding sequence of signal peptide (shown in SEQ ID NO: 19) (i.e., SP, shown in SEQ ID NO: 20), and (5) 3' UTR sequence downstream (shown in SEQ ID NO: 21), and poly-A tail (Poly-A-tail).
  • the in vitro transcription plasmids of the above-mentioned mRNAs were digested with the restriction endonuclease BamHI to be linearized; a conventional DNA purification method was used to purify the mRNAs to obtain in vitro transcription templates; then, based on the templates, in vitro transcription was performed using a T7 RNA in vitro transcription kit (E131-01A, Suzhou Nearshore Protein Technology Co., Ltd.) to obtain in vitro transcribed mRNAs; finally, a lithium chloride recovery kit (S125, Suzhou Nearshore Protein Technology Co., Ltd.) was used to purify the mRNAs by lithium chloride precipitation to obtain purified in vitro transcribed mRNAs.
  • a T7 RNA in vitro transcription kit E131-01A, Suzhou Nearshore Protein Technology Co., Ltd.
  • a lithium chloride recovery kit S125, Suzhou Nearshore Protein Technology Co., Ltd.
  • the capping enzyme kit Cap1 capping enzyme kit (M082-01B, Suzhou Jinan Protein Technology Co., Ltd.) was used to perform 5'-end Cap1 capping on the purified in vitro transcribed mRNA so that it met the conditions for being translated in eukaryotic cells; thereafter, the mRNA was purified again using the same lithium chloride precipitation method as above to obtain purified mRNAs modified by 5'-end capping.
  • HEK293T cells were plated in a 12-well plate and the cell density was about 50% on the next day; 1 ⁇ g of AMAB, AMAB-C140S or AMB-C140S mRNA was added together with TransIT-mRNA reagent (2 ⁇ l) and enhancement reagent (2 ⁇ l) in 100 ⁇ l serum-free Opti-MEM, incubated for 3 minutes, and then added dropwise to the 12-well plate; 36 hours after transfection, the cells were collected.
  • the cells obtained in step (1) were lysed with a cell lysis buffer, and then the cell lysate was mixed with a loading buffer containing dithiothreitol (DTT) and separated by 10% SDS-PAGE; after separation, the membrane was transferred to transfer the protein to a PVDF membrane; then, the PVDF membrane was blocked in 5% skim milk, and the PVDF membrane was incubated with mouse serum immunized with AMAB-C140S as a primary antibody and goat anti-mouse IgG-HRP (EASYBio) as a secondary antibody. Each for 1 hour; finally, use BeyotimeBeyo ECL Plus color development solution to develop color.
  • DTT dithiothreitol
  • FIG. 1 shows the expression of each mRNA in the cells.
  • AMAB, AMAB-C140S or AMB-C140S mRNA can be normally expressed in the cells, and its expression product has only a single band, indicating that it is a single immunogen.
  • Cationic lipids, phosphatidylcholine, cholesterol and PEG lipids were mixed in a ratio of 50:10:38.5:1.5, and then mixed and packaged with the above-mentioned 5'-end capped mRNAs (including “AMAB”, “AMAB-C140S”, “AMB-C140S” chimeric mRNAs, and "A35R”, “M1R”, “B6R” single mRNAs) using the Nanoassemblr Benchtop nanoliposome packaging instrument produced by Precision Nano Systems. After packaging, the buffer solution was replaced with PBS by centrifugation or dialysis. After packaging, the mRNA packaging efficiency was identified using the Quan-iTRibogreen RNA reagent kit from Thermo Fisher, and the packaging efficiency met the standards for mRNA vaccines.
  • animal experiments were performed using female mice of the BALB/c strain aged 6-8 weeks (purchased from Viagra); the experimental groups were divided into an mRNA vaccine immunization group and a negative control group, wherein the mRNA vaccine immunization group included AMAB, AMAB-C140S and AMB-C140S mRNA vaccine immunization groups, and the negative control group was an LNP immunization group.
  • the mRNA vaccine immunization group included AMAB, AMAB-C140S and AMB-C140S mRNA vaccine immunization groups
  • the negative control group was an LNP immunization group.
  • mice in the mRNA vaccine immunization group were immunized with a dose of the chimeric mRNA vaccine prepared in Example 1: AMAB, AMAB-C140S or AMB-C140S mRNA vaccine, or the mRNA mixed vaccine "A/M/B" prepared in Example 1 on day 0 and day 14, respectively; the mice in the negative control group were injected with the same amount of empty LNP at the same time.
  • the vaccination method was intramuscular injection, and the vaccination dose was 7.5 ⁇ g mRNA vaccine or empty LNP per mouse each time.
  • Mouse serum samples were collected on days 13 and 27, respectively, to test the binding antibody titer and pseudovirus neutralizing antibody titer of the immune serum. In addition, mouse spleen samples were collected on day 21 to test T cell immunity.
  • Example 3 Detection of specific binding antibody titers in sera of mice immunized with poxvirus chimeric mRNA vaccine
  • ELISA plates were coated with 0.2 ⁇ g/ml monkeypox virus A35R, M1R or B6R antigen protein (whose amino acid sequences are shown in SEQ ID NO: 1, 3, and 4, respectively), and the coated ELISA plates were blocked in 5% skim milk for 1 hour; then, the serum collected from the mice in each experimental group in Example 2 was incubated at 56° C. for 30 minutes for inactivation; the inactivated serum samples were diluted threefold from 1:200 or 1:1000, and the dilution was added to each well, and then the ELISA plate was incubated at 37° C.
  • monkeypox virus A35R, M1R or B6R antigen protein whose amino acid sequences are shown in SEQ ID NO: 1, 3, and 4, respectively
  • the endpoint titer was defined as the serum dilution factor at which the absorbance produced by the serum (absorbance at 450 nm minus absorbance at 630 nm, as described above) was greater than 2.1 times the background value.
  • Antibody titers below the detection limit were defined as one-third of the detection limit.
  • the antibody titer test results of the sera of immunized mice are shown in Figure 3.
  • the numbers above each bar graph in Figure 3 represent the multiple of the geometric mean compared with the geometric mean of the corresponding antigen in the LNP group.
  • Figure 3 shows that the sera after the first (a) and second immunization (b) of the three mRNA vaccines all contain specific antibodies that can bind to A35, M1 and B6 antigen proteins, among which the antibody titer of the sera after the second immunization is increased by 1 to 2 orders of magnitude compared with the first immunization sera.
  • AMAB, AMAB-C140S or AMB-C140S mRNA vaccines can induce high levels of specific binding antibodies against A35R, M1R and B6R antigen proteins.
  • Vaccinia virus is an orthopoxvirus that can be operated in a biosafety level 2 laboratory and is also a model virus of the genus Orthopoxvirus. Given that VACV has little difference between other orthopoxvirus species, VACV is commonly used internationally to evaluate the cellular and animal levels of orthopoxvirus vaccines. Therefore, we used the vaccinia virus mouse-adapted strain Western Reserve (VACV-WR), which is commonly used internationally, to detect the ability of mRNA vaccines to neutralize live viruses at the cellular level.
  • VACV-WR strain Western Reserve
  • Vero cells were laid in a 12-well plate.
  • the immune mouse serum collected on the 27th day of the immunization procedure in Example 2 was diluted in a deep well plate with 2% FBS DMEM medium, and the diluted serum was diluted 2 times continuously for a total of 10 dilutions.
  • An equal volume of 100 PFU of VACV virus was mixed with the diluted serum and incubated at 37°C for 1 hour.
  • the cell culture supernatant was discarded, and the serum-virus mixture was added and incubated at 37°C for 2 hours.
  • the supernatant was discarded, PBS was rinsed once, and the cells were covered with sodium carboxymethyl cellulose and 2x DMEM 1:1 mixture, and cultured at 37°C for 48 hours.
  • the inhibition rate curve was drawn according to the ratio of the spots under different serum dilution gradients compared with the positive control, and the IC50 (half-inhibitory concentration) value of the curve was used as the PRNT 50 Titer value of the serum sample.
  • the chimeric mRNA vaccine immunization group, the mixed mRNA vaccine immunization group, and the LNP immunization group mice in Example 2 were challenged with VACV-WR intranasal drops on the 29th day after the first immunization, and the challenge dose was 1.9 ⁇ 10 5 PFU.
  • the survival rate and weight changes of mice in the mRNA vaccine immunization group and the negative control group Evaluate the protective effect of each mRNA vaccine on live virus attack in mice.
  • Figure 5 shows that the chimeric mRNA vaccines AMAB, AMAB-C140S and AMB-C140S of the present application can provide 100% protection against VACV-WR-infected mice, and the mice did not show a significant decrease in weight. In contrast, the weight of the mice in the non-immunized group continued to decrease within 4 days until all died.
  • Figure 6 shows that the mRNA mixed vaccine of the A35R peptide, M1R peptide and B6R peptide of the present application (i.e., "A/M/B") can also provide 100% protection against VACV-WR infected mice, and the weight of the mice did not show a significant decrease, or recovered rapidly after a slight decrease. In contrast, the weight of the mice in the non-immunized group continued to decrease within 4 days until all died.
  • the multi-antigen chimeric vaccine of the present application can efficiently stimulate specific immune responses against poxviruses (especially monkeypox virus), and therefore, it is expected to be developed into a preventive and/or therapeutic vaccine product for poxviruses (especially monkeypox virus), and has great clinical application prospects.
  • the poxvirus multi-immunogen chimeric or mixed antigens provided in the present application can stimulate immune responses against two infectious virus particles, intracellular mature virus particles (IMV) and extracellular enveloped virus particles (EEV), thereby efficiently stimulating specific immune protection effects against monkeypox virus; in addition, the poxvirus vaccine of the present application also has good safety, rapid responsiveness and production capacity support, and has excellent clinical application prospects.
  • IMV intracellular mature virus particles
  • EMV extracellular enveloped virus particles
  • SEQ ID NO: 1 amino acid sequence of A35R antigenic fragment I
  • SEQ ID NO:2 amino acid sequence of A35R antigenic fragment II
  • SEQ ID NO:3 amino acid sequence of M1R peptide
  • SEQ ID NO:4 amino acid sequence of B6R peptide
  • SEQ ID NO:5 amino acid sequence of B6R peptide containing C140S mutation
  • SEQ ID NO:6 amino acid sequence of AMAB
  • SEQ ID NO:7 amino acid sequence of AMAB-C140S
  • SEQ ID NO:8 amino acid sequence of AMB
  • SEQ ID NO:9 amino acid sequence of AMB-C140S
  • SEQ ID NO: 10 (DNA sequence of AMAB)
  • SEQ ID NO: 11 (DNA sequence of AMAB-C140S)
  • SEQ ID NO: 12 (DNA sequence of AMB)
  • SEQ ID NO: 13 (DNA sequence of AMB-C140S)
  • SEQ ID NO: 14 (mRNA sequence of AMAB)
  • SEQ ID NO: 15 (mRNA sequence of AMAB-C140S)
  • SEQ ID NO: 16 (mRNA sequence of AMB)
  • SEQ ID NO: 17 (mRNA sequence of AMB-C140S)
  • SEQ ID NO: 19 (signal peptide sequence)
  • SEQ ID NO:20 (coding sequence of signal peptide)
  • SEQ ID NO:22 (DNA sequence of A35R peptide)
  • SEQ ID NO:23 (DNA sequence of M1R peptide)
  • SEQ ID NO:24 (DNA sequence of B6R peptide)
  • SEQ ID NO:25 mRNA sequence of A35R peptide segment
  • SEQ ID NO:26 (mRNA sequence of M1R peptide)
  • SEQ ID NO:27 (mRNA sequence of B6R peptide)

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Abstract

La présente invention concerne un antigène chimérique ou mixte multi-immunogène dirigé contre des poxvirus (en particulier des virus monkeypox), un produit associé de celui-ci, son procédé de préparation et son utilisation. L'antigène chimérique ou mixte selon la présente invention comprend trois immunogènes : (1) une protéine A35R du virus monkeypox ou un fragment antigénique de celle-ci (ou un fragment peptidique dérivé de celle-ci) ; (2) une protéine M1R du virus monkeypox ou un fragment antigénique de celle-ci (ou un fragment peptidique dérivé de celle-ci) ; et (3) une protéine B6R du virus monkeypox ou un fragment antigénique de celle-ci (ou un fragment peptidique dérivé de celle-ci). L'antigène chimérique ou mixte selon la présente invention peut provoquer une réponse immunitaire contre deux particules virales infectieuses de particules virales matures intracellulaires (IMV) et de particules virales enveloppées extracellulaires (EEV), ce qui permet d'obtenir efficacement un effet de protection immunitaire spécifique contre des virus monkeypox ; en outre, le vaccin à poxvirus selon la présente invention a également une bonne sécurité, une réactivité rapide et un support de productivité, et présente de grandes perspectives d'application clinique.
PCT/CN2023/134141 2022-12-02 2023-11-24 Vaccin à poxvirus chimérique multi-antigène et son utilisation WO2024114542A1 (fr)

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